Elderly patients are vulnerable to low cardiac output syndrome after cardiac surgery that obligates global myocardial ischemia and reperfusion (I/R). However, the mechanisms underlying the increased risk of myocardial functional injury in the elderly are incompletely understood. Studies in this proposal will test the hypotheses that aging enhances coronary endothelial response to hypoxic/thermal stress, resulting in augmented secretion of heat shock protein 27 (HSP27, HSP25 in mice), and that extracellular HSP27 functions as an activator of Toll-like receptor 2 (TLR2) and TLR4, and thereby augments post-ischemic injury in aging heart. The hypotheses rest on the following novel findings: 1) in aging humans and mice, heart produces more pro-inflammatory mediators after global I/R that correlate with a greater release of HSP27/25, 2) coronary endothelial cells release HSP27/25 in response to hypoxia, and the release is augmented in endothelial cells from aging hearts; 3) antagonizing extracellular HSP25 suppresses the inflammatory response and improves cardiac functional recovery in aging murine hearts, 4) extracellular HSP27/25 induces the inflammatory response in the myocardium and cardiac cells through a mechanism dependent of both TLR2 and TLR4, and 5) the C-terminal domain of HSP27/25 is critical for its pro-inflammatory effects. The major goals of this proposal are to further determine the effect of aging on the mechanisms of coronary endothelial secretion of HSP27/25, the role of extracellular HSP25 in mediating the hyper-inflammatory response to surgical global I/R in aging hearts, and to elucidate the mechanism by which extracellular HSP27/25 induces the myocardial inflammatory response. We will pursue the following interrelated specific aims: 1) to test the hypothesis that aging augments coronary endothelial response to hypoxic and hypothermic stress for secretion of HSP27/25, 2) to determine the role of extracellular HSP27/25 in the TLR2/4-mediated myocardial inflammatory response in aging hearts, 3) to test the hypothesis that the C-terminal domain of extracellular HSP27/25 activates TLR2 and TLR4 and 4) to explore the therapeutic potential of antagonizing extracellular HSP27/25 and TLR2/4. The proposed studies will provide novel insights into the mechanisms underlying the hyper-inflammatory response of aging heart to surgical global I/R and the basis for developing innovative strategies to protect aging heart during cardiac surgery with obligatory global I/R.